It is demonstrated that for the a-Si/Ag/a-Si (140/30/140 nm) electrode, enhanced capacity (∼59.1%) is derived from the buffer effect and excellent conductivity of silver layer. Silver interlayer may represent a universal platform for relieving stress in a silicon electrode. In addition, its excellent electrical conductivity will decrease the charge transfer resistance of Si electrode for lithium-ion batteries.The laminated construction of an a-Si–Ag thin film electrode is demonstrated, which allows stabilization of the cycling performance of a silicon thin film layer in a lithium-ion battery. A silver thin film plays a determining role in the lithium insertion/extraction process and is incorporated between amorphous Si thin film layers (a-Si/Ag/a-Si), which results in not only high and stable capability, but also the best rate performance compared to that of other electrodes. For the electrode of a-Si/Ag/a-Si, a critical thickness of the silver layer (30 nm) is found; in this case, it exhibits the highest capacity retention of 70% after 200 cycles at a current density of 65.2 μA cm A novel silver interlayer is used to improve the electrochemical performance of the binder-free Si-based thin film anodes. A silicon thin film layer is one of the promising materials for the next-generation LIBs due to its high theoretical capacity (4200 mA h g−1) and low working potential (0–0.5 V vs. Li/Li+).4–7 However, a pristine silicon anode suffers from serious volume change (>300%) during lithium insertion/extraction processes, resulting in electrode pulverization and subsequent loss of electrical contact between the active material and the current collector, eventually leading to poor reversibility and rapid capacity fading.8–10 In addition, its low intrinsic electrical conductivity (10−3 S cm−1) impedes the electrochemical kinetics of the charge/discharge processes, which leads to moderate rate performance.11,12 The disadvantage of silicon thin film layers mentioned above can be addressed by reducing their thickness to a nanoscale size,13 compounding with metals , or using modified collectors.17–20Rechargeable lithium-ion batteries (LIBs) with higher power and energy density are widely used as the most effective energy storage devices, especially with the rapid development of innovative electronic products and electrical vehicles.21–23 Si–Ge,24 Si–Cr,25 Si–Co,26 or Si–Al27, can effectively relieve stress caused by volume changes as well as provide a short pathway for Li+ diffusion and electron transportation.28 In addition, they also display increased electronic conductivity compared to pure silicon.29 Therefore, they can retain a longer lifetime and exhibit more stable rate performances. In addition, silicon-based multilayer thin films directly grown on collectors contain no binders and are conductive, which decreases more than 10 wt% of the inactive materials, thus delivering increased specific capacity.30,31 The preparation processes of electrodes such as coating, drying and pressing are omitted as well. For example, Tobias Placke29 demonstrated that the multilayer Si/C/Si (70/50/70 nm) thin film electrode showed superior cycling performance after 150 cycles with capacity retention of 83 ± 6% compared to a pure Si thin film layer (74 ± 7%) because of the advantage of an additional interlayer that worked as a buffer layer and increased electronic conductivity.Silicon-based multilayer electrodes, such as Si–C,−2) and optimal capacity retention of 80% compared to other electrodes. In particular, the impact of the thickness of the silver layer based on the a-Si/Ag/a-Si structure is investigated. The proportion contributed by the silver thin film layer during cycling is further calculated to comprehend the constitution of the capacity. Furthermore, a major part of this study is assigned to investigate the electronic conductivity as well as mechanical stability during lithium insertion/extraction processes.Herein, a silver thin film layer is used to improve the performance of an a-Si thin film electrode. To study the influence of silver thin film layers on the electrochemical characteristics, Ag/a-Si, a-Si/Ag/a-Si, and a-Si/Ag electrodes are also fabricated by the magnetron sputtering technique. The multilayer system of a-Si/Ag/a-Si exhibits the most stable cycling performance after 150 cycles with the highest discharge areal capacity (111.7 μA h cm2.2.1−4 Pa and Ar (99.999%) was invoked as the working gas. The Si thin films were deposited at a constant radio frequency power supply of 100 W. Ag thin films were deposited by direct current sputtering at an applied power of 40 W. In both cases, for Si and Ag thin films, the sputter processes were performed at room temperature with a working pressure of 1 Pa. The deposition rates calculated from the total film thickness were 4.67 nm min−1 for silicon and 30.0 nm min−1 for silver. To eliminate impurities present on the target surface, both silicon and silver targets were pre-sputtered for more than 10 min.Silicon and silver materials were grown on copper foil in a magnetron sputtering system (FJL560III-type). The base pressure in the sputtering chamber was approximately 3 × 102.2+ laser (514.5 nm) as the excitation source at room temperature. The X-ray photoelectron spectroscopy (XPS) analysis was performed on a K-Alpha+ instrument from Thermo fisher Scientific using monochromatic Al Kα radiation  and high energy electron flooding for charge compensation.The morphologies of the as-produced Si–Ag samples were characterized by field emission electron microscopy . Structural characterization was performed by X-ray diffraction  operated at 35 kV and using Cu-Kα radiation (1.5406 Å) in the range of 5–90°. The micro-Raman spectra were recorded using backscattering geometry with Ar6 in a mixture of ethylene carbonate (EC) and dimethyl carbonate (DMC) (1 : 1 in volume ratio).The electrochemical properties of the Si–Ag thin films directly deposited on Cu foil (14 mm diameter and 9 μm thickness) were tested using 2032-type half-cells in an argon-filled glove box. The assembled cells contained Si–Ag thin film as the working electrode, an electrolyte, a separator (Celgard 2325) and a lithium metal foil as the counter electrode. The electrolyte was 1 M LiPFvs. Li/Li+) at a scan rate of 0.1 mV s−1 and EIS measurements were obtained in the frequency range from 100 kHz to 0.01 Hz after 5 cycles. The cycled samples were disassembled in the glove box and cleaned separately with acetone and ethanol and dried naturally.Galvanostatic charge–discharge and rate performance tests were carried out on a battery testing system (Neware BTS3000) between 0.01 V and 1.5 V. An electrochemical workstation (chi 760e) was used to perform cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). CV was performed for the first 5 cycles in the potential range of 0.01–1.5 V (3.n-(100) single crystalline silicon substrates. The pure amorphous silicon layer was about 280 nm in size (sub). About 30 nm Ag thin film was deposited on the surface of pure Si layer to protect the susceptible Si electrode/electrolyte interface, and the rigid structure could limit the volume expansion of silicon during the process of lithiation and delithiation . In addition, no indication of the crystalline silicon phase is shown since no apparent peak at 520 cm−1 is revealed.18 To further illustrate the existence of silver and silicon phases, the most representative electrode of a-Si/Ag/a-Si deposited on Cu foils is tested using XRD  is employed to evaluate the ability of Li-ions’ insertion/extraction in the anodes fabricated in this study, and it is defined as the total discharge/charge capacity (μA h) divided by the projected surface area (π × 7 mm × 7 mm ≈ 1.54 cm2) of the composite electrodes.41 It can be seen that the pure a-Si electrode delivers the first discharge capacity of 173.2 μA h cm−2 with an initial coulombic efficiency (CE) of 70.6% and displays the best cyclic stability for 70 cycles without a clear capacity loss. However, the discharge capacity degrades rapidly down to 70.2 μA h cm−2 after 150 cycles, indicating only 53.5% retention of the initial reversible capacity  and cycling CE (84.2%), but the capacity degrades rapidly after 115 cycles, which is similar to that observed for pure silicon electrode. During 1 to 150 cycles, for the a-Si/Ag/a-Si electrode, there is no observable sharp decline in capacity. It is clear that the a-Si/Ag/a-Si electrode possesses the most stable cycling performance with the highest discharge areal capacity (111.7 μA h cm−2) and optimal capacity retention of 80% after 150 cycles compared to other electrodes. Among all the materials, a-Si/Ag as the anode material shows the worst cycling performance, which indicates that the silver thin film deposited on the copper foil cannot increase the adhesion of silicon to the substrates such as Ti22,23,42 and Cr.43–45 The CE values after 150 cycles are 98.3%, 95.2%, 99.5% and 99.1% for the electrodes of a-Si, Ag/a-Si, a-Si/Ag/a-Si, and a-Si/Ag, respectively. Also, 30 nm-thick Ag layers as anodes were tested for comparison.To demonstrate the effect of the position of the deposited silver thin film layer, the cycling performances of the Ag, a-Si, Ag/a-Si, a-Si/Ag/a-Si and a-Si/Ag electrodes were measured at the same current density of 65.2 μA cmrocesses . The arecapacity . The poo1,46It is therefore suggested that the capacities of Ag/a-Si and a-Si/Ag/a-Si anodes are not the sum of the capacities of silver and silicon electrodes but a greater improvement thereof. Slight increase in the capacities for the a-Si and a-Si–Ag electrodes in the initial several cycles is linked to the gradual activation of the silicon host, which may result from the gradual infiltration of the electrolyte.1Charge/discharge voltage profiles for pure a-Si, Ag/a-Si and a-Si/Ag/a-Si electrodes are depicted in −1 of pure a-Si thin film (a), Ag/a-Si (b), and a-Si/Ag/a-Si (c) electrodes are depicted. There are two cathodic peaks and two anodic peaks in all CV measurements, indicating the two-step lithiation as well as two-step delithiation processes, which is in good agreement with the charge/discharge voltage profiles  layer should occur between 0.5 and 1 V, and it is usually indicated by a peak in the CV data around 0.8 V due to the dissociation of electrolyte.ensities . When th results  and 4, t−2). This is because the difference in current densities between two adjacent cycles is small. When the current density was switched from 261.0 μA cm−2 back to 65.2 μA cm−2, the capacities of all samples increased rapidly. For the a-Si/Ag/a-Si electrode, the capacity value increased from 89.2 to 134.8 μA h cm−2, corresponding to the 50th and 51st cycles. This huge capacity change was caused by the large change in current density. A similar phenomenon of the poor rate dependence can be found in previous research as well.11,55–57Rate dependence is poor in all samples at different cycles, especially at the first five current densities  or incorporated between the amorphous Si thin film layers (a-Si/Ag/a-Si) improves the structural stability to survive the drastic volume change and the huge mechanical stress during cycling.Rs is the ohmic series resistance associated with the cell components such as the electrolyte or other components,59 and RCT is the charge transfer resistance corresponding to the diameter of the semicircle at high frequency; W is the Warburg impedance related to the Li ion diffusion into the active material, which is generally identified by a sloping straight line. The capacitor components are replaced by constant phase elements (CPE).11,46 The diameters of the semicircle for the Ag/a-Si  and a-Si/Ag/a-Si  electrodes are significantly smaller than that of the pure Si  electrode, revealing lower charge-transfer impedances. It is indicated that the introduction of a silver film can enhance the conductivity of a silicon thin film. It is also recognised that Si-based electrodes generally exhibit cracking and crumbling during lithium insertion and extraction. After the recycling process, partial broken silver films in Ag/a-Si and a-Si/Ag/a-Si electrodes, converted into fillers form and reinserted these cracks. This restructured morphology, as a conductive percolation network, enhanced the cycling performance of Si-based electrodes. Therefore, the silver thin film located on the surface of or in the middle of the silicon film can enhance the stability of Si-based electrodes during the charge/discharge processes. In our study, the Ag film as an embedded layer plays two main roles, especially in the multilayer a-Si/Ag/a-Si structure. On the one hand, the Ag layer is ductile enough to relieve the stress caused by large volume changes owing to Li alloying and dealloying reactions with Si. On the other hand, Ag has attracted great attention due to its excellent electric conductivity and its negligible effects on the lithium storage behavior of Si.57The cycling performances of pure a-Si, Ag/a-Si, and a-Si/Ag/a-Si thin film electrodes are further studied by electrochemical impedance spectroscopy (EIS) measurements  after 5 st to the 200th cycle compared to a pure Si thin film. The initial reversible discharge areal capacities are 162.8, 146.4, 139.6, and 150.6 μA h cm−2 and the discharge areal capacities decay to 84.9, 91.6, 98.1, and 89.7 μA h cm−2 after 200 cycles, indicating the capacity retentions of 52.2%, 62.6%, 70.3%, and 59.6% for 5 nm Ag, 15 nm Ag, 30 nm Ag, and 60 nm Ag, respectively (It has been mentioned above that silver thin film layers incorporated between the amorphous Si thin film layers (a-Si/Ag/a-Si) can enhance the cycling performance of the silicon thin film electrode. In this section, the a-Si/Ag/a-Si electrode was selected to study the optimum thickness of the Ag layer as well as the influence of the Ag layers on the electrochemical performance. The cycling performances of the a-Si/Ag/a-Si electrodes with different thicknesses of silver layers  are compared. For better comparability, the cycling data of a pure amorphous Si thin film (280 nm) are also depicted in ectively . The pur41 in the corresponding cycles by referring to the following equation:n is the cycle number from 2 to 200; Ca-Si/Ag/a-Si(n) and Ca-Si(n) stand for the discharge capacities of a-Si/Ag/a-Si and pure a-Si electrodes, respectively, in the corresponding cycle. For the a-Si/Ag/a-Si electrode, CAg retains a value of more than 58 μA h cm−2 even after 200 cycles. To comprehend the contribution to the total capacity, the proportion contributed by the silver thin film layer during cycling is given by the following equation:The contribution of the silver thin film is mainly studied based on the a-Si/Ag/a-Si (140/30/140 nm) electrode. The capacities contributed by the Ag thin film electrode can be approximately obtained by carrying out the subtraction calculationPAg(n) shows a slight declining trend before 60 cycles; then, it increases notably up to 59.1% in the remainder of the 200 cycles. The above variation in tendency is due to the interaction of a-Si/Ag/a-Si and pure a-Si thin film electrode cycles. The intercalated a-Si/Ag/a-Si structure is equivalent to reducing the thickness of a single a-Si film layer by forming two a-Si film layers with the same thickness of 140 nm. Previous research13,21,58 results show that a thinner a-Si film layer exhibits more stable cycling performance and the protective behavior of a silver film also improves the electrochemical kinetics by restraining the volume expansion to avoid mechanical breakage.+/electron transport in multilayer a-Si/Ag/a-Si electrodes, electrochemical impedance spectra (EIS) of a-Si/Ag/a-Si electrodes with different layer thicknesses of silver  after the first five cycles were obtained, as shown in RCT value decreased along with increasing silver content because the silver nanoparticles provided a favorable environment for the migration of lithium ions into/from the silicon particles.11 However, this is not the case for the multilayer structured electrodes. In our study, the RCT values of the a-Si/Ag/a-Si electrodes measured with different layer thicknesses of silver  are 110, 290, 118, and 60.5 Ω, respectively, as shown in RCT values of the multilayer a-Si/Ag/a-Si electrodes decrease with an increase in the thickness of the silver layer except for the silver layer thickness of 5 nm. The above results are explained by the interaction of silver content, the mutual mixing degree and the crack size of the Si-based film during the lithium insertion/extraction processes and the mechanism is explained below.To further study the mechanism of Li11 When the silver content is abundant, other factors play a minor role. Thus, the value of RCT tends to decrease when the thickness of the silver layer increases from 15 to 60 nm. On the other hand, silicon-based multilayered anode materials will intermix after cycling,29 which also leads to lower RCT. Because of the severe volume change, Si-based film electrodes tend to crack and pulverize during cycling. Meanwhile, a part of the broken silver layer or particles is converted to fillers and enters the cracks, which results in a conductive percolation network in the Si-based electrodes, as shown in RCT value than the a-Si/Ag/a-Si electrodes with silver layer thickness of 15 nm.On the one hand, increasing the silver content can effectively enhance the electron conductivity of the silicon-based electrode.From the observations discussed above, we can confirm that the multilayer a-Si/Ag/a-Si electrode exhibits very excellent electrochemical performance compared to the pure silicon film electrode. This positive influence can be ascribed to two reasons: first, the additional interlayer of ductile silver acts as a buffer layer as it is a compact layer and experiences only slight volume expansion, which leads to smaller crack sizes after cycling. Second, the broken silver layer acts as a conductive agent between the cracks of the silicon film. Silver nanosheets can connect the broken inactive silicon film portions together and convert them back into an active substance. Therefore, the multilayer a-Si/Ag/a-Si electrode has higher areal capacity after 200 cycles and the charge transfer resistance decreases along with increasing silver content.4.−2 and optimal capacity retention of 80% after 150 cycles at 65.2 μA cm−2 compared to other electrodes), but also exhibits the best rate performance compared to other electrodes. It also possesses lower charge-transfer impedances due to excellent electron conductivity of silver. The structure of the a-Si/Ag film as the electrode material shows the worst cycling performance, which indicates that the silver thin film deposited on the copper foil cannot increase the adhesion of silicon to the substrate. The Ag thin film layer deposited on the surface of an a-Si layer (Ag/a-Si) significantly improves the electrochemical performance in terms of an enhanced coulombic efficiency because the surface layer of silver can effectively protect the highly reactive Si surface from the bulk electrolyte. In these studies, the influence of the Ag thin film layer thickness was also examined. With the increase in the thickness of silver film, the capacity retention increases first and then decreases. A critical thickness of 30 nm is found for the silver thin film layer, at which the highest capacity retention (70% after 200 cycles) can be achieved. This study provides a way to improve the electrochemical performance of a silicon thin film layer electrode with an additional silver layer as the interlayer (a-Si/Ag/a-Si).In this study, Ag/a-Si, a-Si/Ag/a-Si, and a-Si/Ag thin film layer electrodes carefully designed and fabricated by the magnetron sputtering method to stabilize the cycling performance of the silicon thin film layer electrodes were thoroughly investigated. The results show that the silver thin film incorporated between the amorphous Si thin film layers (a-Si/Ag/a-Si) not only displays high and stable capability (with the highest discharge areal capacity of 111.7 μA h cmThere are no conflicts to declare.


Cell uptake in L6 cells by 18F-FCH was performed in a complete medium containing serum  and a serum-free medium . Small-animal-dedicated PET/CT imaging with 18F-FCH was examined in in-vivo models with rats that were starved for 2 days to cause muscle atrophy. After the hind limbs were dissected, starvation-induced in-vivo models were anatomically confirmed by reverse-transcription polymerase chain reaction to evaluate the expression levels of the atrophy markers muscle RING-finger protein-1 (MuRF-1) and atrogin-1. 18F-FCH uptake was lower in the starvation-induced cells than in the untreated group, and in-vivo PET uptake also revealed a similar tendency  = 0.26 ± 0.06 versus 0.37 ± 0.07, respectively). Furthermore, the expression levels of MuRF-1 and atrogin-1 mRNA were significantly increased in the starvation-induced muscle atrophy of rats compared to the untreated group. 18F-FCH PET/CT may be a promising tool for diagnosing skeletal muscle atrophy.Imaging techniques for diagnosing muscle atrophy and sarcopenia remain insufficient, although various advanced diagnostic methods have been established. We explored the feasibility of  The most common body changes that occur with aging are gradual and generalized skeletal muscle disorders that involve an accelerated loss of muscle mass and function . RecentlMuscle mass is regulated by protein synthesis and degradation . Muscle 18F-FCH PET/CT, taken up into cells by choline transporters and subsequently phosphorylated by choline kinase, is mainly used for the detection, staging, and surveillance of malignancies, such as lung and prostate cancers [18F-FCH for the diagnosis of muscle atrophy has not been studied and needs to be verified in various aspects.Choline is an essential component of cell membrane phospholipids in skeletal muscle cells ,11. It b cancers ,17,18. H18F-FCH uptake, using both in-vivo and in-vitro models of starvation-induced muscle atrophy, and explored the possibility of its use as a diagnostic biomarker. Based on the results of this analysis, we aimed to provide basic data for diagnosing muscle atrophy via 18F-FCH PET in the skeletal muscle.Recently, to achieve the diagnosis of sarcopenia, studies on using imaging modalities, dual-energy X-ray absorptiometry (DXA), computed tomography (CT), magnetic resonance (MR), and ultrasound (US), have been reported ,20,21. H5 cells/well), as previously described [2. After 24 h, the cells were switched to serum-free medium to induce starvation. Cell morphology was observed using light microscopy, and cell size was examined using ImageJ software version 1.52a . L6 cells were cultured in 6-well plates (1 × 10escribed . The celn = 6) and the group starved for 48 h . The ST group was provided with free access to water during the period of starvation, whereas the UN group was maintained under standard conditions. The UN group was fasted for 4 h prior to the 18F-FCH injection. Before the PET acquisition, the weight of the UN group was 298.0 ± 4.66 g and the ST group was 234.9 ± 5.64 g.Sprague–Dawley rats  were purchased from Orient Bio  and maintained in a controlled environment . To allow them to adapt, the rats were housed individually in ventilated cages for 1 week with free access to water and food. Prior to the experiments, the rats were randomly divided into two groups, namely, the untreated group  using a disposable dedicated 18F-FCH cassette and a reagent kit. The isolated product with non-decay-corrected radiochemical yield, calculated from trapped radioactivity on a QMA cartridge, was 26.7 ± 1.4% (n = 11), with over 99% radiochemical purity as determined by radio-thin liquid chromatography at the end of the synthesis.18F-FCH. Each group of cells (UN and ST) was subdivided into three subgroups. For each treatment (UN and ST), a subgroup was incubated for either 30, 60, or 120 min under the same conditions described until trypsinization. The collected cells were measured for the radioactivity of 18F-FCH uptake using a 2480 Wizard2 automatic gamma counter , automatically corrected by the half-life of fluorine-18.Cells were cultured by dividing them into two groups, one cultured in complete medium containing serum  and the other in serum-free medium (starved group). After 24 h, the medium in each group was replaced with the same medium in which it was incubated with the exception that it contained 2/O2 and all rats in each group were administered a single dose of 18F-FCH (14.8 ± 2.96 MBq) intravenously, following which PET images were acquired for the diagnosis of muscle atrophy in the untreated and starved rats. After 40 min of conscious radiotracer uptake, rats were anesthetized with 2.5% isoflurane in a 7:3 mixture of N2/O2, and sequential PET-CT scans were acquired for 20 min using a dedicated small animal PET/CT scanner . CT scans were used for attenuation correction and anatomical localization of the PET signals. The acquired PET images were reconstructed using the three-dimensional Adjoint Monte Carlo method with scatter and random corrections. The volume of interest (VOI) in both legs (hindlimb) was delineated by the intensely visualized region in the summed image. VOIs were drawn on CT images of individual animals in a slice-by-slice manner. Regional uptake of radioactivity was decay-corrected to the injection time and expressed as the average standardized uptake value (SUV), which was normalized to the amount of radioactivity injected and the animal’s body weight. InterView Fusion software  was used to analyze the data of the SUV in the VOIs after reconstruction and quantification [The rats were anesthetized with 2–3% isoflurane in a 7:3 mixture of Nfication .n = 3). The muscles were fixed in 4% paraformaldehyde, embedded in paraffin, and serially sectioned into 5 μm slices. The prepared sections were cleared with xylene and hydrated with an ethanol gradient . To analyze the histological changes, the sections were stained with Masson’s trichrome (MT) solution. The tissues were captured using a microscope  at 200 × magnification. Muscle fiber areas were analyzed using ImageJ version 1.52a .Histochemistry was performed as described previously . BrieflyThe expression level of choline acetyltransferase (ChAT) was analyzed using immunohistochemistry as previously described . The tisThe mRNA levels were analyzed as previously reported . The gasp < 0.05.All statistical analyses were performed using Prism software . Quantitative data are expressed as mean ± standard deviation, and comparisons of quantitative data between the two groups were analyzed using an unpaired t-test. Statistical significance was set at 18F-FCH cell uptake experiment were analyzed in the untreated and starved groups. For in-vivo validation, small-animal-dedicated PET/CT with 18F-FCH, and ChAT expression assays were performed.To test the hypothesis that tracking changes in choline uptake can confirm the diagnosis of muscle atrophy, we performed in-vitro and in-vivo experiments. For the in-vitro experiments, the cell size changes of L6 cells under a microscope and Skeletal muscles undergo atrophy during starvation. In fact, L6 cell size decreased and lengthened after 24 h of serum starvation A.18F-FCH cell uptake to determine whether muscle atrophy could induce choline uptake. As shown in 18F-FCH uptake compared to the UN group .Anatomically, the soleus muscle is surrounded by the gastrocnemius. In fasting-induced muscle atrophy in rats, the soleus muscles showed a significantly reduced mass by about 20%, followed by the plantaris and gastrocnemius muscles . p < 0.001).As evaluated by 18F-FCH PET/CT could be used as a radiotracer for imaging-based diagnosis of muscle atrophy in vitro and in vivo. Through in-vitro analysis, we observed lower 18F-FCH uptake in starved L6 cells and reduced uptake in the starvation-induced muscle atrophy of rats. As a result, in the case of muscle atrophy induction, changes in the mRNA expression levels of fat and carbohydrate metabolism were evident, and the gene expression levels of MuRF-1 and atrogin-1 increased. A morphologically altered pattern indicated muscle atrophy. Therefore, these preliminary data suggest this method could be a diagnostic tool for muscle atrophy using 18F-FCH PET/CT.In the present study, we explored whether 18F-FCH radiotracer is involved in lipid metabolism and is significant in atrophy modeling, in vitro and in vivo. Therefore, we suggest that 18F-FCH PECT/CT is a possible molecular imaging diagnostic tool for muscle atrophy.The normal physiological condition of the skeletal muscle and its regulation of muscle protein synthesis/breakdown may be altered due to various pathological conditions, such as cachexia, heart disease, obesity, and aging. Human skeletal muscle reaches its final cell number early in life but decreases in number and size around the age of 30 years . The mec18F-FCH radiotracer. There was a higher utilization of fat metabolism compared to carbohydrate use, but this is hypothesized to be due to a rapid decrease in overall energy metabolism. The skeletal muscle has the greatest plasticity in any organ of the human body. An increase in skeletal muscle leads to an increase in basal metabolic rate, and an increase in skeletal muscle decreases energy consumption [Choline plays an important role in maintaining cellular integrity and in the synthesis of neurotransmitters. It plays a role in overall metabolism, as it is involved in cell membrane signaling, fatty acid transmission, brain function improvement, and smooth neurotransmission through methyl group metabolism ,29,30. Isumption . Accordisumption . This is18F-FCH radiotracer. In muscle atrophy, metabolic function is significantly reduced, and the demand for choline increases due to a lack of protein or fat metabolism [18F-FCH uptake. In particular, in image acquisition within 60 min, 18F-FCH PET/CT showed the tracking of muscle loss. One of the limitations of our study is that it is difficult to distinguish different muscle types, soleus, plantaris, and gastrocnemius, in preclinical studies. Another is that the extent of the quantitative decrease in muscle fiber area . Therefore, we cautiously expect that 18F-FCH PET will be clinically useful in the diagnosis of atrophy. Furthermore, since 18F-FCH PET is routinely used for the diagnosis of prostate cancer in clinical practice, it has the potential to be easily used in human clinical trials for diagnosing muscular atrophy. This method may also be beneficial to study muscle atrophy conditions for which muscle biopsies are not feasible .To answer this question, we performed a PET/CT imaging study using tabolism . Cholinetabolism . In our ber area B and theber area A are notIn addition, we also identified the intensity of ChAT, the enzyme used to synthesize acetylcholine. Diamond et al. assessed the role of muscle mass and function in regulating ChAT activity in mice, postulating that ChAT activity could be an ideal indicator for biochemical measurement of the response to activation or inactivation signals in muscle . In this18F-FCH PET/CT as a potential diagnostic tool for muscle atrophy. In addition, fasting-induced muscle atrophy has been shown to activate MuRF-1 and atrogin-1 and alter the amount of choline in the body. Since our study was focused on molecular imaging, at the cellular and animal levels, its application in other models of muscle atrophy needs to be studied further. In conclusion, we propose


The major modification of in-plane magnetic anisotropy of the Co2MnSi thin film is controlled obviously by the piezo-voltages of the lead zirconate titanate (PZT) piezotransducer, accompanied by the coercivity field and magnetocrystalline anisotropy significantly manipulated. Because in-plane cubic magnetic anisotropy and uniaxial magnetic anisotropy coexist in the Co2MnSi thin film, the initial double easy axes of cubic split to an easiest axis (square loop) and an easier axis (two-step loop). While the stress direction is parallel to the [1−10] easiest axis (sample I), the square loop of the [1−10] direction could transform to a two-step loop under the negative piezo-voltages (compressed state). At the same time, the initial two-step loop of the [110] axis simultaneously changes to a square loop (the easiest axis). Otherwise, we designed and fabricated the sample II in which the PZT stress is parallel to the [110] two-step axis. The phenomenon of VCMA was also obtained along the [110] and [1−10] directions. However, the manipulated results of sample II were in contrast to those of the sample I under the piezo-voltages. Thus, an effective dual-axis regulation of the in-plane magnetization rotation was demonstrated in this work. Such a finding proposes a more optimized method for the magnetic logic gates and memories based on voltage-controlled magnetic anisotropy in the future.Voltage controlled magnetic anisotropy (VCMA) has been considered as an effective method in traditional magnetic devices with lower power consumption. In this article, we have investigated the dual-axis control of magnetic anisotropy in Co 2MnSi/GaAs/PZT heterostructures under piezostrain. The periodic response of magnetization is achieved under the pulsed piezo-voltages of ±40 V.Dual-axis controlled magnetic anisotropy and magnetization 90° rotation are demonstrated in Co The magneto-crystalline anisotropy is controlled by voltage through the piezo-voltage-induced strain transformed to the magnetic thin film. Usually, the change of magnetocrystalline anisotropies is related to the strain-manipulated variations of the lattice constant.23 However, the previous relative studies of strain-controlled magnetization rotation were mainly demonstrated in a uniaxial regulation manner.22–24,26 The stress regulation characteristic disappears or weakens when the specific crystal orientation of magnetic thin films rotates relative to the stress axis. This problem would be effectively avoided by realizing dual-axis or multi-axis stress-regulated magnetization rotation. However, the related research is still relatively lacking.Pure electrical manipulation of magnetization rotation in magnetic devices is a desirable way for spintronic applications, which is suitable for the scaling of devices in the integrated circuit. To date, there have been multiple types of electrical manipulation ways of the magnetization rotation or magnetic anisotropy variation, such as spin–orbit torque (SOT),27 which are promising candidates for the next generation information processing and storage in spintronic devices. The coexistence of the in-plane cubic and uniaxial magnetic anisotropies was observed when Heusler alloys are deposited on the GaAs (001) substrate.28–30 The initial in-plane easiest axis (square loop) and easier axis (two-step loop) have been measured along two axes of minimum value of the cubic anisotropy, which is caused by the superposition of the uniaxial anisotropy. It is well known that a uniaxial stress could induce an extra uniaxial anisotropy in magnetic films. Thus, using the competition of uniaxial anisotropy induced by interface and stress in magnetic films can realize the regulation of magnetic anisotropy energy and magnetization rotation.In recent years, the Co-based full-Heusler alloys have attracted considerable interest due to the high spin polarization and Curie temperature,2MnSi/GaAs/PZT heterostructures through piezo-voltage-induced strain. By studying the variation of the magnetic coercivity (Hc) and remnant magnetization (Mr) in Co2MnSi magnetic thin film, the strong voltage-controlled magnetic anisotropy was verified. Furthermore, we measured the periodic-strain controlled magnetization by applying pulsed piezo-voltages. Two stable states have been achieved with the periodic measurement in two samples. The dual-axis control of the magnetic anisotropy in this work proposes a method of voltage-controlled magnetic logic devices, which will simplify the growth process of magnetic materials and reduce energy consumption.In this work, we have studied the dual-axis control of magnetic anisotropy in the Co2MnSi thin film was grown at 250 °C on a GaAs (001) substrate using molecular-beam epitaxy (MBE).28,29 Its Curie temperature is 985 K.27 Following the growth of 10 nm-thick single crystal Co2MnSi, a 3 nm thick platinum (Pt) layer was deposited to avoid the oxidation  were measured by longitudinal magneto-optical Kerr microscopy (Nano MOKE3) and a superconducting quantum interference device (SQUID) magnetometer. The piezo-voltages were applied with an Agilent B1500A with the leading and trailing time being both 100 ns. All the measurements were carried out at room temperature.The 10 nm Co2MnSi/GaAs/PZT heterostructures controlled by piezo-voltage-induced strain. Two samples of Co2MnSi/GaAs are bonded to the PZT, where the [1−10] and [110] crystal orientations are parallel to the z-axis in samples I and II, respectively. The piezoelectric ceramic blocks are stacked along the z direction, and the reverse piezoelectric effect appears in the d33 mode . When the positive and negative piezo-voltages (UPZT) are applied, the PZT would exhibit a tensile and compressive strain, respectively.23 Under the stress regulation, the samples I and II will undergo different regulation effects. 2MnSi thin film along the [110], [1−10] and [100] directions. The saturation magnetization Ms is about 884 emu cm−3. The [1−10] orientation is an easy axis with a square loop and the [110] orientation is a two-step loop with Mr = 0 when the magnetic field is zero. The [100] orientation is a hard axis with a 400 Oe saturation field  to [110] (UPZT = −40 V) direction. For sample II, the values of KU and KC are also obtained to be −0.91 ± 0.05 kJ m−3 (0.77 ± 0.04 kJ m−3) and 12.42 ± 0.62 kJ m−3 (18.62 ± 0.93 kJ m−3) under a piezo-voltage of 40 V (−40 V), which also induced the transformation of the easiest magnetization axis and magnetization 90° rotation.Through the demonstration of dual-axis control of magnetic anisotropy in epitaxial Co2MnSi/GaAs/PZT heterostructures, it will be promising to be applied in the design of magnetic functional devices, such as magnetic tunneling junction (MTJ) and planar Hall devices. To further study the response performance of the Co2MnSi/GaAs/PZT device, we measured the magnetization during the periodic change of piezo-voltage between −40 and 40 V in sample I without the external magnetic field. The magnetization periodically switched from 7.5 to 2.3 mdeg, which correspond to the ‘1’ and ‘0’ states of the logic device  control of magnetic anisotropy in epitaxial CoB. Z. and C. L. conceived the work. S. M. performed the sample growth. B. Z. fabricated the devices and performed the experiments. B. Z., C. L., P. H. and Z. H. analyzed the data. B. Z., C. L. J. Z. and Z. H. wrote the manuscript. All authors discussed the results and commented on the manuscript.There are no conflicts to declare.


Background and Objectives: In order to accelerate the risk stratification of patients referred to the Emergency Department (ED) with interstitial pneumonia, it could be useful to provide new and effective laboratory tests for use. The aim of our study was to evaluate the prognostic role of two biomarkers, bio-adrenomedullin (Bio-ADM) and proenkephalin (penKid), in patients with interstitial pneumonia (IP) at ED admission. Materials and Methods: In 153 consecutive patients with IP, both from COVID-19 or non-COVID-19 etiology, we measured, in a prospective observational manner, penKid and Bio-ADM at ED admission and after 24 h. In order to evaluate patient outcomes, 30-day follow-ups were also performed. The endpoints were 24 h, 10-day, and 30-day mortality. Results: Both biomarkers were shown to be good predictors of adverse events at 30 days, with Bio-ADM outperforming penKid. Bio-ADM was linked with 24 h and 10-day patient mortality. Moreover, PenKid was related to parameters defining worsening kidney function. Conclusions: Both in patients with COVID-19 or non-COVID-19 interstitial pneumonia at ED admission, Bio-ADM and penKid were good predictors of patient mortality. To evaluate these two biomarkers could be considered to be useful during the first evaluation in the ED when integrated with clinical scores. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the novel coronavirus that causes coronavirus disease 2019 (COVID-19). The first cases of COVID-19 were reported in the Chinese city of Wuhan located in the province of Hubei in December 2019 .COVID-19-infected cases present with symptoms such as fever, dry cough, and other flu-like findings, but with a possible rapid deterioration with lower airway involvement and complicated by pneumonia ,4. In thBiologically active adrenomedullin (Bio-ADM) is an emerging biomarker of endothelial function and vascular integrity already proven to be of utility in patients with sepsis in the ED. Plasma adrenomedullin is associated with short-term mortality and vasopressor requirement in patients admitted with sepsis .Bio-ADM could serve as a useful and objective biomarker to predict severity, organ failure, and 30-day mortality in septic patients ,12,13. HProenkephalin (penKid) is a promising biomarker that has been studied in critically ill patients which may play a role in the evolution of COVID-19 infection in a life-threatening disease and which has never been studied yet in the context of interstitial pneumonia . The bioThe aim of this study was to investigate the prognostic role in short-term mortality of Bio-ADM and penKid in ED patients affected by interstitial pneumonia caused by COVID-19 in comparison with patients with interstitial pneumonia of any other cause.This monocentric, prospective observational study was conducted in the ED of San Giovanni Addolorata Hospital on behalf of the postgraduate medical school of Emergency Medicine of the University Sapienza of Rome. The study was approved by the institutional Research Ethics Committee “Lazio 2” . After informed consent was signed, we enrolled 153 patients who were admitted to the emergency ward from April 2020 to April 2021.-Age ≥ 18 years;-First-presentation symptoms such as dyspnea, fever, and dry cough;-High-Resolution Computed Tomography (HRCT) scan findings of suspicious images for interstitial pneumonia;-Adhesion to informed consent.Inclusion criteria:-Age < 18 years;-Refusal of consent;-Dyspnea from a non-infectious cause;-Symptom onset more than 3 days before the enrollment.Exclusion criteria:The HRCT was considered suspect for interstitial lung involvement if more than 25% of the lung parenchyma was involved or if ground glass, crazy paving, architectural distortion, honeycombing, or consolidations were found in more than two lung fields ,20,21.All of the patients enrolled underwent two SARS-CoV-2 oro-pharyngeal swabs within 24 h of each other to confirm or exclude SARS-CoV-2 infection. We used real-time polymerase chain reaction (RT-PCR) molecular tests and TaqMan-based detection .Enrolled patients were evaluated on ED admission (T0), after 24 h (T1), and then 30 days later (T30) with a telephone call follow-up.At T0, a SARS-CoV-2 swab, lung HRCT, routine blood tests, and arterial blood gas analysis (ABG) were performed on each enrolled patient. Subjects who met the enrollment criteria were assigned to one of the two groups, COVID+ or COVID−, depending on whether they were, respectively, positive or negative on the swab. An additional blood sample was withdrawn through a vacutainer system with EthylenDiaminoTetracyc Acid (EDTA) and stored at −20 °C in a freezer for further analysis of Bio-ADM and penKid. Clinical history before admission to the ED, existing therapies, drug allergies, ongoing symptoms, radiological findings, and hemodynamic parameters were also recorded at this time. qSOFA and SOFA scores were recorded at T0 and recalculated at T1 to stratify patients’ mortality risk.At T1, patients underwent a new clinical evaluation and qSOFA and SOFA were recalculated and recorded. Patients negative on the first oropharyngeal swap underwent a second test to confirm the negative results and, in case of positivity, were switched to the COVID+ group. We used RT-PCR molecular tests and TaqMan-based detection .A second sample in an EDTA-containing vacutainer for Bio-ADM and penKid measurements, as well as another ABG, was obtained for all patients at this time.At T30, patients were contacted by telephone for follow-up. The onset of any potential complications, new hospitalization, or death in the previous 30 days was recorded. Attention was paid to whether complications arose in the first 10 days after ED admission (T10) or in the following 30 days (T30).Both T0 and T1 venous samples were centrifuged within one hour in San Giovanni Addolorata Hospital with a swinging bucket centrifuge set at 1200 rpm for 10 min at 36 °C. We then obtained two 0.5 mL centrifuged samples and stored them at −20 °C. The obtained samples were shipped to Sphingotec GmbH laboratories in Germany for the analysis of Bio-ADM and penKid concentrations.®Bio-ADM®) developed by Sphingotec GmbH . Bio-ADM immunoassay is a one-step sandwich chemiluminescence immunoassay based on acridinium NHS-ester labeling for the detection of human adrenomedullin (ADM) in unprocessed, neat plasma. It uses two mouse monoclonal antibodies, one directed against the mid-region and the other directed against the amidated C-terminal moiety of ADM. The assay uses 50 µL of plasma samples/calibrators and 220 µL of labeled detection antibody. Once thawed back to room temperature, Bio-ADM in EDTA plasma is stable for up to 24 h, and samples are unaffected by at least up to four freeze–thaw cycles. This assay is highly specific to Bio-ADM as it only reacts with the mature amidated C-terminus of ADM and not with other variants of the precursor of ADM (Pro-ADM). Pro-ADM was measured using a sandwich ELISA on a Luminex®platform  [Plasma Bio-ADM was measured using an immunoassay  .PenKid was measured in duplicate using a chemiluminescence immunoassay , as described: 2 mouse monoclonal anti-PenK antibodies were developed by immunization with PENK peptide . One antibody (2 mg) was used to coat polystyrene tubes. The other antibody labeled with methylacridinium ester served as the detector antibody. Standards  and samples (50 mL) were incubated in tubes with the detector antibody (150 mL). After equilibration, the tubes were washed, and bound chemiluminescence was detected with a luminometer .The patients were divided into two groups (COVID− and COVID+) according to the results of the swab. The statistical significance was determined with a threshold of 95% (α < 0.05). The whole statistical examination has been performed using the statistical free software R . Continuous variables were tested for normality with the Shapiro–Wilk test and summarized as mean and standard deviation (SD).The differences between the means of the different groups were tested using a single-way ANOVA and Tukey’s test for post hoc analysis. Categorical variables were summarized in crosstab, expressed as a% of the own group, and analyzed using a χ2 test. If the test gave a significant result, that was further analyzed with a z-test.The box-plot graphs were realized for the main results in order to visualize the values of distribution. A survival analysis was performed in order to evaluate the predictive power of Bio-ADM and penKid. The cut-off for the variables utilized for the analysis was 100 pg/mL and 43 pmol/L, respectively, for penKid and Bio-ADM, as previously described by Lundberg OHM et al.  and MariThe differences in survival time between the groups were graphically compared by constructing Kaplan–Maier curves and verified with a log-rank test.p-value = 0.2).We enrolled 153 patients : 101 (66%) affected by COVID-19 pneumonia (COVID+) and 52 (34%) affected by interstitial pneumonia not related to SARS-CoV-2 (COVID−). The mean age of the entire population was 70 ±17 years, with no differences between COVID+ and COVID− .p-value 0.053) and 19% vs. 5% for minor complications .A successful rate of response was obtained in 109 patients (71%) upon follow-up and the percentage of dropout was the same in the two groups. Among the 44 patients for whom we were unable to obtain follow-up, there were some who were homeless, some who were foreigners in transit in Rome, and some patients for whom the address we collected was no longer available at the time of the call. Fifteen patients (9.8%) of the entire studied cohort died within 30 days. Two deaths occurred in the first 24 h. Six patients died within the first 10 days. The number of deaths increased by another seven units at 30 days. Most deaths, 12 cases, occurred in the COVID+ group (11.9%), compared to COVID−, in which only three patients died (5.8%); however, the difference did not reach statistical significance. Both major and minor complications were more common in the COVID+ group compared to COVID-: respectively, 19.6% vs. 5.8% for major complications .p-value = 0.9). Hb and pH appeared higher in the COVID+ group  than in COVID− , with a slight but not significant reduction in Lac in the COVID+ group with respect to COVID− . These results are consistent with the worst oxygen delivery in the COVID− group at the time of presentation at the ED. In COVID+, there was a different cellular peripheral inflammatory response with a reduced white blood cell (WBC) count compared to COVID−  and an increased neutrophil concentration compared to COVID− . Platelet count was also reduced in the COVID+ group compared to COVID− . No other significant differences were found between the other laboratory tests.No changes in blood gas exchanges were found between the two pulmonary interstitial infections with similar P/F values between COVID+ (359 ± 168) and COVID− , T10 , and T30 . The mean values of Bio-ADM at T0 were 184 ± 98 pg/mL for those who died within the first 24 h, 101 ± 71 pg/mL for those who died in the first 10 days, and 73 ± 60 pg/mL in patients who died at the 30th day of follow up. The respective values were in the group of survivors at T1: 37 ± 31 pg/mL; for the survivors at T10: 36 ± 29 pg/mL; and in the group of survivors at T30: 36 ± 30 pg/mL. These results are shown in Bio-ADM values strongly correlated with mortality at T1 .No relationship was found between Bio-ADM values and the development of complications both major and minor. In the first group of patients, the mean ± SD was 43.03 ± 45.63 pg/mL, and in the patients that developed complications, this value was 49.4 ± 32.89 pg/m .We performed a Cox analysis, including values of Bio-ADM and PenKid over or below the cutoff at T0 in order to verify their Hazard Ratio (HR). We also decided to forcefully add age, creatinine value at T0, and COVID-19-positive diagnosis in the analysis, considering these variables as potentially predictive of mortality. The complete model results significantly differ from the null model (As shown in p 0.02) for Bio-ADM > 43 pg/mL and an HR of 1.09  for age.With an HR of 2.95 .As can be seen from p-value of 0.0001.We observed a positive linear regression between PenKid levels at T0 and creatinine at T0 with an R of 0.55 and a p-value of 0.003 but an R of only 0.29 .A correlation analysis between AKI and PenKid > 100 mmol/L was performed, and it showed a slight positive correlation with a Neither Bio-ADM nor penKid appear to be influenced in any way by the etiology of lung infection and no differences were observed in biomarker predictivity in the COVID + and COVID− groups.The main finding of our study has been the predictive mortality value of Bio-ADM and penKid, measured in the ED in patients presenting with interstitial pneumonia both related and not related to COVID-19 infection. Furthermore, Bio-ADM seems to correlate mostly with short-term mortality , while penKid correlates better with 30-day mortality.In the absence of specific treatment strategies for COVID-19, there is an urgent call for clear guidance by early risk-stratifying biomarkers . In our p-value 0.0004) in patients with interstitial pneumonia. According to the currently available data, there is no study proving a relationship between Bio-ADM values at ED admission and 24 h mortality in patients with interstitial or lobar pneumonia, sepsis, or septic shock. The earliest mortality studied so far is at 4 days [p-value of 0.0003 and 0.0032, respectively). These results are comparable to those that have already been obtained in various studies already published in the literature which establish how Bio-ADM values higher than the cut-off correlate with mortality in septic patients [p-value < 0.0001). We may suppose that since the SOFA score assesses sepsis-related organ failure and sepsis is usually determined by bacteria, it was more likely for a patient suffering from non-COVID pneumonia to develop sepsis.As far as we know, this is the first study proving a strong correlation between admission Bio-ADM levels and mortality within 24 h  is a humanized monoclonal antibody targeting the N-terminus of ADM ,31,32, aIn the last few years, many studies concerning penKid have been carried out. Our objective was to evaluate the biomarker’s diagnostic and prognostic role as a predictive factor of AKI . It is kMany studies addressing the diagnostic and prognostic role of penKid in various types of patients both in the ED and ICU are available in the literature. It has been proven that elevated blood penKid values at admission to the ICU are associated with organ failure occurring within the first 2 days, they correlate with kidney dysfunction, and they are good predictors of 30-day mortality regardless of the disease leading to the hospitalization. Even though enrollment criteria were different, our study set in the ED confirmed this biomarker’s role in predicting renal impairment, and the early detection of this marker at admission to the ED could allow the early start of a therapy that optimizes renal function, perhaps reducing the onset of irreversible damage or disability. The fact that penKid predicts mainly 30-day mortality may be due to the fact that this biomarker is a good predictor of disease sequelae; this kind of predictive power has already been evidenced in a previous retrospective trial conducted on patients admitted to the ICU for many reasons ranging from sepsis, cardiac arrest, or trauma .Our study shows that in patients presenting to the ED with either COVID+ or COVID− interstitial pneumonia, Bio-ADM seems to be a strong predictor of 24 h, 10-day, and 30-day mortality. PenKid strongly relates to AKI and mortality at 30 days.Therefore, it seems that the usage of these two biomarkers in the setting of the ED for prompt risk stratification could lead to a better and faster decision-making process and could improve the prognosis of this kind of patient.


It was found that human miRNA-146a and miRNA34b/c are important microRNAs and are functioning either as onco-miRNAs, or acting as tumor suppressors, in different conditions. To date, no study has been performed to evaluate the alterations of miRNA-146ars2910164 and miRNA34b/crs4938723 polymorphism as a risk factor in the development of thyroid cancer in the Pakistani population. Mutational analysis of rs2910164 and rs4938723 of miRNA-146a and miRNA-34b/c was carried out to check their association with the development of thyroid carcinogenesis. Material and Methods: Papillary thyroid cancer (PTC) patients with age and gender-matched controls were recruited for the present study. DNA extraction, genotyping of rs2910164 and rs4938723 was carried out by ARMS-PCR. Statistical analyses were carried out using SPSS software (version 20). Results: The odds ratio for risk allele C of rs2910164 for patients and controls was 23.0168 (3.0321–174.7208) with a p-value of <0.0001, showing that the frequency of the major allele G was lower in patients while the frequency of minor allele C was higher in patients. Similarly, the odds ratio for risk allele C of rs4938723 was 1.8621 (1.0321–3.3596) with a p-value of <0.03788 showing significant association with the development of thyroid cancer. Conclusions: The study highlights the significant association of miRNAs SNPs as one of the genetic risk factor for PTC. It was concluded that miRNA-146a (rs2910164) showed higher frequency of minor allele C in patients. Similarly in miRNA-34b/c gene SNP rs4938723 was observed to have a strong association with the development of thyroid cancer as the frequency of rare allele C was higher in patients.Background: Rationale: The  Differentiated thyroid carcinoma constitutes about 90% of thyroid cancers, includes papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC) . Papillae genome . miRNA-1ntiation . The seqogenesis . Pre-miRrs2910164 (miRNA-146a) played a key role in genetic predisposition to PTC through regulation of different other miRNAs [rs2910164 is playing a key role in transferring the benign nodules into PTCs [rs2910164 with PTC.This important miRNA Single Nucleotide Polymorphism SNP has been studied in PTC and demonstrated to be related to the spread of cancers like prostatic adenocarcinoma and gastric cancer . Jazdzewr miRNAs , but latnto PTCs . It was nto PTCs . This strs4938723 of miRNA-34b/c. Many pieces of evidence have shown that miRNA-34b/c plays a role in the development of different types of cancer. It was found that in the development of thyroid cancer miRNA-34b/c has an altered expression and contributes to carcinogenesis [pri-miRNA-34b/c is the reason for the development of cancer. It was revealed that the variation of rs4938723 C to T may affect the binding of the GATA-X transcription factor which then causes the altered expressions and results in the development of carcinomas [miRNA-34 , and two different types of primary miRNAs are encoding these three miRNAs. Pri-miRNA-34b/c encodes the transcripts of miRNA-34b and miRNA-34c while miRNA-34a is encoded by its own transcript [Another aspect of this study was the mutational analysis of rcinomas . p53 is anscript .miRNA-34b/c have a major role in the development of tumors and alterations of these two have a connection with alterations of miRNA-34b/c rs4938723 that result in the development of thyroid cancer [miRNA34b/crs4938723 polymorphism as a risk factor in the development of thyroid cancer in Pakistan. For this purpose, mutational analysis was carried out to check its association with thyroid carcinogenesis in the Pakistani population.The p53 is considered as the “guardian of the genome” which shows how much this gene is important in cell cycle control, apoptosis, and maintenance of DNA integrity . It was d cancer . To datemiR-34b/c rs4938723 CT/CC genotypes were associated with a significantly high risk of hepatocellular cancer [miRNA-34b/c rs4938723 CC genotype decreased the risk of colorectal cancer in a Chinese population at about 0.56-fold [An association study has reported that the r cancer . Resultsr cancer . Conflicr cancer . Gao et .56-fold . In the Two hundred and five confirmed papillary thyroid cancer (PTC) patients with age and gender-matched normal controls were selected for the current study. Demographical and clinical data of the study subjects were obtained on a structured questionnaire. Blood samples were obtained and collected in EDTA coated tubes and stored at low temperature (4 °C). Informed consent statements were signed by patients and healthy individuals. The study protocol was approved by the ethical review committee of PMAS Arid Agriculture University, Rawalpindi and from the hospitals of Rawalpindi and Islamabad, Pakistan (3 October 2018).All those patients who were confirmed as thyroid cancer patients by the oncologists were included in the study while all patients who were having other problems of the neck or mentioned as hypothyroid or hyperthyroid patients were excluded from the study.rs2910164 of miRNA-146a primers were designed by primer blast. Selected primers were forward primers, F-inner-5′-CATGGGTTGTGTCAGTGTCAGACGTG-3′, forward-outer F outer-5′-TAGACCTGGTACTAGGAAGCAGCTGCAT-3′-F and reverse primers R-inner-5′-GATATCCCAGCTGAAGAACTGAATTTGAG-3′reverse outer R-outer-5′-ATACCTTCAGAGCCTGAGACTCTGCCTT-3′-R. Primers were also designed for the selected sequence of DNA for miRNA-34b/c rs4938723 by primer blast. For this purpose, the following forward and reverse primers were designed. Forward inner F-inner-5′-CCTCTGGGAACCTTCTTTGACCTCTC-3′-F, F-outer-5′-CTCCCAGAAGTCCTCTGTAACTGTCCCT-3′-F and reverse primer, R-inner-5′-AGAAGGGAGGTCCTCAATGAGAGCTTTA-3′-R, R-outer-5′-TAGTCAAATAGTGAGCCAGGCAGCTTGT-3′-R were designed for miRNA-34b/c rs4938723.The experiments were carried out using tetra ARMS-PCR. For mutational analysis of Taq polymerase enzyme, 1 μL of 25 mM MgCl2, 1 μL of 2 mM dNTPs  and PCR water 12.7 μL/sample. The PCR product was analyzed by 2% w/v agarose gel electrophoresis. The statistical analysis of data was carried out by SPSS (version 20). p-value < 0.05 was considered statistically significant for the T-test. Graphs were constructed by using graph pad prism software.PCR was carried out in a total volume of 25 μL having 5 μL of genomic DNA (20 ng), 2.5 μL 10× PCR buffer (NH4)2SO4, 1 μL of 20 nM forward and reverse primers each, 0.3 μL of 5 U/μL The patients were categorized based on their age and gender into different groups. Most of the patients in the present study were females having a frequency of about 86% while males were only 14%. Patients were categorized into three different age groups, i.e., later age (more than 60), middle-age (40–60), and early age (below 40). It was seen that all the patients were having papillary thyroid cancer with its different subtypes .rs2910164 SNP of miRNA-146a with the risk of thyroid cancer progression was determined with the help of tetra ARMS-PCR. Hardy–Weinberg calculations for patients’ samples showed that the observed frequencies of homozygous wild type GG, heterozygous CG, and homozygous recessive CC were 60 percent, 38.5 percent, 1.5 percent while the expected frequencies are 62.8056%, 32.888% and 4.3056%, respectively. The observed frequencies of GG, CG, and CC in controls were 98%, 2% and zero% while the expected frequencies were 98.0%, 1.98% and 0.01% respectively. The p-value for the difference between the observed and expected frequencies in patients was 0.95, (>0.05) and is statistically non-significant, indicating that there is no significant difference between the expected and observed frequencies of genotypes in the patients. The Chi-square value was 2.9109 for patients. The p-value for controls was 0.69 (>0.05), which is also statistically non-significant, indicating no significant difference of observed and expected frequencies in the control, with a Chi-square value of 0.012. It was seen that the rare genotype CC is significantly associated with the risk of development of thyroid cancer with a p-value 0.00001 (<0.05) (Association of  (<0.05) .miRNA-34b/c with the risk of thyroid cancer development was also determined with the help of tetra ARMS-PCR. Hardy–Weinberg calculations for patient samples showed that the observed frequencies of homozygous wild type TT, heterozygous TC, and homozygous recessive CC were 10 percent, 36 percent and 54 percent while the expected frequencies are 8.1225%, 40.755%, and 51.1225%, respectively. The observed frequencies of TT, TC, and CC in controls were 30%, 54% and 15% while the expected frequencies were 32.8182%, 48.3636% and 17.8182%, respectively. The p-value for the difference between the observed and expected frequencies in patients was 0.654129 (>0.05), and is statistically non-significant, indicating that there is no significant difference between the expected and observed frequencies of genotypes in patients. The Chi-square value was =0.8489 for patients. The p-value for controls was 0.510533 (>0.05), which is also statistically non-significant indicating no significant difference of observed and expected frequencies in the control; the Chi-square value was 1.3446. It was also observed that the rare genotype CC is significantly associated with the risk of development of thyroid cancer with a p-value 2 × 10−8 (<0.05) (The relationship of the SNP of  (<0.05) .rs2910164. The frequency of homozygous dominant GG was 60% and 98% in patients and controls, respectively, while the combined frequency of heterozygous and homozygous mutant CG/CC was 40% and 2% in patients and controls, respectively. The p-value for the genotypes frequency in patients and control was 0.00001 (<0.05), with a chi-square value of 43.52. The p-value was statistically significant (<0.05) indicating that the combined effect of genotypes and allele frequency showed statistically significant involvement with the development of disease. The combined effect of genotype frequencies in patients and the control group was also calculated for rs4938723. The frequency of homozygous dominant TT was 10% and 30% in patients and controls, respectively, while the combined frequency of heterozygous and homozygous mutant TC/CC was 90% and 68% in patients and controls, respectively. The p-value for the genotypes frequency in patients and control was 0.00030426 (<0.05), with a chi-square value of 13.044. The p-value was statistically significant (<0.05) indicating that the combined effect of genotypes and allele frequencies are significantly involved in the development of disease  with a p-value < 0.0001 (<0.05). The odds ratio for heterozygous CG genotype was 30.6748 (7.1467–131.6616) with a p-value < 0.0001 at a 95% confidence interval. The odds ratio for the homozygous recessive genotype CC was 3.0833 (1.2649–7.5157) with the p-value of 0.010255 at 95% CI  with a p-value of <0.0001. These results showed that the frequency of the major allele G was lower in patients while the frequency of minor allele C was higher in patients  with a patients .rs4938723 in the current study. The odds ratio for homozygous dominant genotype TT was 0.2222 (0.1012–0.488) with a p-value < 0.0001 (<0.05). The odds ratio for heterozygous CG genotype were 0.4792 (0.2718–0.8447) with p-value < 0.010488 at 95% confidence interval. The odds ratio for the homozygous recessive genotype CC was 6.6522 (3.3862–13.0683) with the p-value < 0.0001 at 95% CI. Forest plot also showed that the genotype CC is associated with the disease group  and p-value was <0.05474, while the odds ratio for risk allele C was 1.8621 (1.0321–3.3596) with a p-value of <0.03788. These results showed that the frequency of the major allele T was lower in patients while the frequency of minor allele C was higher in patients  and a Chi-square was 30.178 showing a statistically significant and strong association of the mutant allele with thyroid cancer risk in the patient group as compared to normal controls  and a Chi-square value of 3.922. There was a statistically significant and strong association of the mutant allele with thyroid cancer risk in the patient group as compared to normal controls (Allele frequencies for controls .rs2910164) of miRNA-146a, which is associated with the risk of papillary thyroid cancer and also affects miRNA-146a processing. It was found that heterozygous genotype GC is associated with a high risk of papillary thyroid carcinoma while homozygous GG or CC genotypes have not shown any association when cancerous tissues were compared with normal tissues [rs2910164, which causes the change from a G:U pair to a C:U mismatch in the basic structure of the mir-146a precursor. This substitution changes the specific activities of mature hsa-mir-146a while binding to its specific targets, which results in increased expression of hsa-mir-146a [miRNA-146a. MITF is a factor that is a protooncogenic transcription factor and it acts as a master regulator in the development of melanocyte functioning and survival. It was also implicated in choroidal melanoma pigmentation and proliferation [rs493872 significantly increased the risk of PTC [miRNA-34b/c play a significant role in the development of thyroid cancer.Jazdzewski et al. found that different polymorphisms affect the expression of miRNA and, in turn, this affects the mRNA maturation or recognition which may be an important risk factor in the susceptibility to disease. A variant was described in the passenger strand of the pre-miRNA rs210164 of feration . A studyferation . Data frk of PTC . Granja k of PTC . Similark of PTC . A furthmiRNA-146a rs2910164 and miR-34b/c rs4938723 is associated with the risk of thyroid cancer in the studied population. Further, these studies also indicated that these miRNAs can be used for prognostic and diagnostic purposes in future for the early detection and treatment of thyroid cancer patients.Statistical analysis in the present study indicated for the first time that the genetic variability of the


The COVID-19 pandemic continues to be a worldwide health issue. Among hemodialysis (HD) patients, two-dose immunization schemes with mRNA vaccines have contributed to preventing severe COVID-19 cases; however, some have not produced a sufficient humoral response, and most have developed a rapid decline in antibody levels over the months following vaccination. This observational, prospective, multi-center study evaluated the humoral response in terms of presence and levels of IgG antibodies to the receptor-binding domain of the S1 spike antigen of SARS-CoV-2 (anti-S1-RBD IgG) to the third dose of SARS-CoV-2 mRNA vaccines, either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer), in 153 patients from three dialysis units affiliated to Hospital Clínic of Barcelona (Spain). Most hemodialysis patients responded intensely to this third vaccine dose, achieving the seroconversion in three out of four non- or weak responders to two doses. Moreover, 96.1% maintained the upper limit or generated higher titers than after the second. BNT162b2 vaccine, active cancer, and immunosuppressive treatment were related to a worse humoral response. Every hemodialysis patient should be administered a third vaccine dose six months after receiving the second one. Despite the lack of data, immunosuppressed patients and those with active cancer may benefit from more frequent vaccine boosters. The coronavirus disease 2019 (COVID-19) pandemic has been a worldwide health issue, and though cases have risen and fallen during its several waves , it undoubtedly continues to affect us to this day . In thatFrom the many vaccines available for use in the general population, some authors have suggested using the mRNA vaccines for special immune-deficient individuals, such as those receiving hemodialysis, because of their better immunogenicity results . This isCurrently, only two mRNA vaccines are approved for clinical use. These are the mRNA-1273 (Moderna) and the BNT162b2 (Pfizer-BioNTech). Among in-center HD patients, current two-dose immunization schemes with any of these mRNA vaccines have contributed to preventing severe COVID-19 cases in most patients up to six months after completing vaccination , humoralIn light of these data and after the pandemic’s fifth wave, Spain approved administering a third mRNA vaccine dose in HD patients. Since then, several studies have reported a successful experience in the humoral response to a third vaccine dose in most HD patients ,19,20,21This study aims to determine the humoral response in HD patients, especially those who did not respond to the standard two-dose vaccination scheme, and to assess the increase in anti-S1-RBD IgG levels after the third dose of a SARS-CoV-2 mRNA vaccine. Centre de Diàlisi i Recerca Aplicada Clínic and Institut Hemodiàlisi Barcelona. Every patient received a complete scheme of three doses with the same vaccine type  following the national health authorities’ instruction: high-risk patient profiles such as HD ones who had not been infected in the previous 30 days received a booster. More information regarding the allocation process and other methodological aspects can be found in a previously published study, as this study represents the continuation of the follow-up with a cohort of 201 patients who had already received a two-dose vaccination scheme during February 2020 [This is an observational, prospective, multi-center study designed to evaluate the immunogenicity of the third dose of SARS-CoV-2 mRNA vaccines, mRNA-1273  and BNT162b2 , in HD patients from Hospital Clínic of Barcelona and two affiliated centers, ary 2020 .All prevalent adult patients on a maintenance HD program in the three mentioned dialysis facilities were considered for inclusion. Patients were excluded if they had been vaccinated in other healthcare centers, did not receive vaccination because of refusal, were admitted to the hospital during the inclusion period, had a SARS-CoV-2 infection 30 days before the third dose administration, or refused to participate in the study. Included participants were classified according to their previous response status, i.e., non- or weak responders if their anti-S1-RBD IgG levels were inferior to 150 U/mL, and responders if they were equal or superior to this specified amount. The humoral response to the third dose was measured two weeks after the vaccine administration with the Siemens Healthineers Atellica® IM SARS-CoV-2 IgG (sCOVG) assay, which detects anti-S1-RBD IgG. The assay is considered non-reactive when the result is less than 1 or reactive when greater than or equal to 1, with a maximum measurable range of up to 150 U/mL limiting the antibody response intensity analysis and constituting a potential source of bias. According to the manufacturer, this test has a 96.41% sensitivity (95% CI 92.74–98.54%) and 99.9% specificity (95% CI: 99.63–99.99%).Other studied variables included were age, gender, dialysis vintage, and comorbidities such as body mass index, diabetes, active cancer, patients with human immunodeficiency (HIV), hepatitis B (HBV) or C (HCV) chronic viral infections, previous SARS-CoV-2 infection, or the use of immunosuppressive therapy. Before initiating the vaccination program, these demographic and medical history data were collected from electronic medical records.The primary outcome evaluated in this study was the qualitative humoral response to the complete three mRNA vaccine doses. In that sense, seroconversion was used to name those patients whose anti-S1-RBD IgG levels resulted in greater than or equal to 1 in a previous negative person. Secondary evaluated outcomes were quantitative anti-S1-RBD IgG levels among different groups of patients to evaluate the response to the third booster compared to the previous two-dose scheme.p-value inferior to 0.05 was considered statistically significant. Analyses were performed with IBM SPSS® Statistics 26th version (https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-26 accessed on 11 January 2022) and the graphics with GraphPad Prism® 8th version (https://www.graphpad.com/scientific-software/prism/ accessed on 11 January 2022).Quantitative variables are described as mean and standard deviation, while qualitative variables are reported as absolute and relative frequencies. Univariate analysis was used to estimate the associations between vaccination and outcomes. Differences in qualitative variables were analyzed with the χ2 test or the Fisher’s exact test when one or more expected values were less than five or the data were very unequally distributed among the table’s cells. The normal distribution in the quantitative variables was tested with the Shapiro–Wilk test and Q–Q plots. The quantitative variables’ analysis between the two groups was conducted with the Mann–Whitney U test when non-normal or the independent Student’s t-test when normally distributed. For analyses between more than two groups, related-samples Cochran’s Q test or related-samples Friedman’s two-way analysis of variance by ranks with Bonferroni corrections were performed if variables were qualitative or quantitative, respectively. A two-sided All patients provided their written informed consent to participate in this study. The study was conducted following the World Medical Association Declaration of Helsinki, national and local laws, and good clinical practice standards. The institute’s committee on human research approved it. The authors have no conflicts of interest to declare regarding this work. This work has neither received public nor private funding for its implementation.The humoral response to the third dose of mRNA SARS-CoV-2 vaccine was assessed in the 153 included HD patients. Among them, 84 (53.6%) were male and had a median age of 72.12 ± 14.44 years. Regarding the vaccine label allocation, 71 (46.4%) received three doses of BNT162b2 and 82 (53.6%) of mRNA-1273. Among the forty-eight patients who were lost during follow-up between the previously mentioned study and this one, twenty died, sixteen received a kidney transplant, four were vaccinated outside the HD center, four refused to receive the third dose, three were transferred to another HD center, and the remaining one was on vacation when blood tests were obtainedp = 0.338) showed a positive anti-S1-RBD IgG test. Percentages of positive serologies to each dose can be seen in In total, 149 patients (97.4%) out of the 153 vaccinated individuals seroconverted after the complete three-dose scheme. The clinical characteristics of the four patients (2.6%) who did not respond are seen in p < 0.001) and much less than the 88.9% after the first dose (p < 0.001), as seen in Although there were only 4 non-responders, there were 19 other patients with a weak response, for a total of 23 patients (15% of the cohort). These results are inferior to the 60.8% of non- or weak responders reported after the second dose  of these non- or weak responders to the second dose presented a sufficient response to the third one. p < 0.001). When comparing by vaccine type, although those receiving mRNA-1273 did not have significantly lower anti-S1-RBD IgG levels  at baseline, after each vaccine dose the differences significantly increased in favor of the mRNA-1273 vaccine . Anti-S1-RBD IgG mean levels increased after each mRNA vaccine dose received by this population. At baseline, the measured mean antibody levels were 3.76 ± 18.08 U/mL, which then increased after the first dose to 21.79 ± 47.31, to 97.79 ± 58.88 U/mL after the second, and to 134.35 ± 41.06 U/mL after the booster with the third . All comWhen comparing antibody levels from the second and third doses, we found that 60 (39.2%) patients maintained the same intensity, with anti-S1-RBD IgG higher than 150 U/mL, 87 (56.9%) had higher levels after the third dose than the second, and there were 6 (3.9%) patients whose levels were lower after the third dose than after the second.p < 0.001) and after the first two vaccine doses . However, after the third dose, this difference lost statistical significance , probably due to a lack of quantification of antibody titers over 150 U/mL.We compared the 20 patients (13.1%) who had a previous COVID-19 infection and found that their anti-S1-RBD IgG levels were higher at baseline , but also to have a lower proportion of non- or weak responders. However, these results must be regarded with caution, as in data from previously published articles, we found that these differences disappeared at least 3 and 6 months after the second dose of vaccine 8.8%, but.Regarding other immunosuppressed populations, a recently published study evaluated the humoral response in hemato-oncological patients to two doses of the BNT162b2 vaccine . This coAnother immunodeficient population is elderly individuals; however, there are scarce data regarding three-dose studies from this group. Research on their response to two doses has found age to be unrelated to the intensity of the humoral response, though an association was found with diabetes mellitus, cancer, and previous SARS-CoV-2 infection . This loOur study has some limitations. Firstly, our laboratory was only able to report anti-S1-RBD IgG levels up to 150 U/mL. This could mean that a difference may exist on higher ranges that we have not measured, which would be in accordance with that reported in other works . SecondlMost hemodialysis patients responded intensely to this third vaccine dose, achieving the seroconversion in three out of four non- or weak responders to two doses. Moreover, 96.1% maintained the upper limit or generated higher titers than after the second one. Therefore, we recommend every hemodialysis patient receives a third vaccine dose six months after receiving the second one. Further studies are needed to evaluate the maintenance of these immune responses and the potential need for more booster doses in every HD patient or specific subpopulations. Promising data on a fourth dose in kidney transplant recipients are being gathered, and a fifth one is being scheduled, as they have a poor response and worse outcomes than their peers who remain on dialysis.


The aim of the study was to examine the efficacy and debris extrusion of XP -Endo Finisher and XP-Endo Finisher R when used in the removal of root canal filling as supplementary files.Sixty single-rooted mandibular premolars with single canals were selected. After root canal preparation and obturation, roots were distributed across four groups according to the method of retreatment (n=15): H files, D Race files, D Race +XP-Endo Finisher and D Race+ XP -Endo Finisher R. After retreatment completion, the debris was dried in a hot air oven and weighed. Later, the coronal, middle and apical thirds were assessed using the stereomicroscope. One-way ANOVA followed by Tukey’s post hoc test was used to compare across all tested groups. The significance level was set at 0.05 (P<0.05).XP- Endo Finisher R exhibited significantly cleaner root canals than XP- Endo Finisher (41.58±10.56 and 52.68±9.94 respectively) and extruded more debris apically (16.56%±4.07 and 12.82%±3.41 respectively) (P<0.001).Although none of the tested approaches rendered root canals free of filling remnants, the XP-Endo Finisher R cleaned canals significantly more than the XP-Endo Finisher and extruded more debris apically. Proper mechanical preparation is crucial to regain access to the apical foramen. This enables irrigants to reach and disinfect the root canal space before sealing all portals of exits .Recently, a new instrument, XP-Endo Finisher file  was introduced to the market. It is a # 25 tip non-tapered rotary Nickel Titanium (NiTi) instrument made of a special alloy  .The XP-Endo Finisher R  is a new variant of the XP-endo Finisher file. It is designed for retreatment cases, according to the manufacturer, and the file has a bigger core diameter (tip # 30), and changes in the instrument tip, making it stiffer and thus more aggressive. The goal of these adjustments is to improve its ability to touch and remove root filling components that remain after traditional retreatment procedures . A studyFurther challenges can occur during the root canal retreatment including the apical extrusion of dentine chips, gutta-percha, remaining pulp tissues, and microorganisms . These eTo date, no published research has investigated the quantity of the apical extruded debris during an endodontic retreatment procedure using XP-Endo Finisher and XP-Endo Finisher R as supplementary files. Our null hypothesis was that there is no significant difference between the tested techniques in cleaning efficacy and apical debris extrusion during retreatment procedures.The present study was started after the approval of the Research Ethics Committee at the College of Dentistry, Suez Canal University (registration no. 216/2019).Based on previous studies, sample size calculation was performed using G*Power version 3.1.9.2. The effect size was 0.57 using an alpha (α) level of 0.05 and a beta (β) level of 0.05, i.e., power=95%; the estimated minimum sample size (n) was 60 teeth for the four groups in this study, 15 teeth per group .Sixty human mandibular premolars were only used to standardize the canal morphology. They were freshly extracted for periodontal or orthodontic purposes. After viewing the teeth using bidirectional radiographic images displaying buccolingual and mesiodistal directions. The study only included teeth having a single, straight root canal (10°) according to the Schneider method . Teeth oThe following inclusion criteria were applied to the samples: 1) free from root caries, 2) single canals type I Vertucci's classification with mature apex, 3) no internal resorption or calcification, 4) no previous endodontic treatment, and 5) and no signs of cracks.Teeth were immersed in 2.5% sodium hypochlorite for 2 hours to enable disinfection .The remaining external tissue fragments and calculus were removed from the external surface of teeth by ultrasonic scaling. Teeth were then kept in saline until use. All samples were decoronated using a high speed diamond bur and copious amount of coolant . DecoronRoots were placed in wet gauze to avoid dehydration. K file # 10  was introduced to the root canal and moved down gently until it was seen from the major apical foramen. Then, subtraction of 1 mm to obtain the working length (WL). Samples were prepared with Bio-Race instruments  BR0 25/ 0.08, BR1 15/ 0.05, BR2 25 /0.04, BR3 25 /0.06 and BR4 35/ 0.04 via gentle pecking motion to the WL. The endodontic motor  was adjusted according to the Bio-Race manufacturer’s instructions at a speed of 600 rpm and torque of 1 Ncm. Flute cleaning was completed after each file removal from the root canal .At each instrument, the canals were irrigated with 1 mL 2.5% NaOCl  using a NaviTip 30-G needle . The needle was 2 mm away from the WL. After root canal preparation, the samples were irrigated using 17% EDTA  activated for one minute. IrriSafe instrument  25/21 mm was mounted onto an ultrasonic unit  for passive ultrasonic irrigation. Later, rinsing was performed with 2.5% NaOCl, and then distilled water .Lateral compaction was utilized in obturation after dryness of the root canal with 35/ 0.04 paper point . AH plus sealer , was mixed according to the manufacturer’s instructions. The tip of Gutta-Percha cone # 35/0.04  was lightly coated with AH plus and gently introduced into the canal. Then finger spreader, # 25/ 0.02 , was inserted in the canal rotated and withdrawn. Accessory Gutta-Percha cones # 25/ 0.02 coated with thin layer of AH plus were placed into the canal until there was no more space for accessory cones beyond 2-3 mm in the canal. After root canal filling was completed, a periapical radiographic image was taken for evaluation .After root canal treatment completion, access cavity was sealed by Coltosol F temporary filling . Teeth were stored at 37°C in a 100% humidity incubator  for 7 days .One of the co-authors, who was not involved in the steps of retreatment, performed the blind allocation of the teeth. This allocation was completed via Microsoft Excel randomization completed in groups. Roots were equally divided and randomly placed into 4 groups according to the instruments used in retreatment (n=15).Root canal filling in the coronal and middle thirds was removed using Gates-Glidden burs  # 4 and 3 in combination with hand H files via the crown down technique. The filling mass was penetrated by # 60 H file followed by 55, 50, 45 and 40 until the WL was regained with file # 35. The apical third was enlarged to a # 40 K hand file .Retreatment was performed using D-Race . The coronal third and beginning of the middle third of the root filling were removed using the DR1 instrument 30/0.10 at a speed of 1000 rpm and a torque of 1.5 Ncm. The DR2 instrument 25/ 0.04 was used at a speed of 600 rpm and a torque of 1 Ncm until the working length was maintained. Apical preparation was attained by Bio-Race instruments BR3 25/ 0.06, BR4 35/ 0.04 and BR5 40/ 0.04 at a speed of 600 rpm and a torque of 1 Ncm.For both groups 1and 2, to keep patency of the canal, K file #10 was used to confirm canal path before reintroducing the next file. Moreover, after each instrument, 2.5% NaOCl with 1ml volume was utilized. Upon withdrawal, the files were cleaned of any obturating material before being reintroduced into the root canal. Retreatment procedures were continued until no gutta-percha remnants were observed on the files.Retreatment was performed with D-Race files as described in group 2. In addition, XP–Endo Finisher file was used as a supplementary file following the retreatment procedures. The XP-Endo Finisher file was used according to the manufacturer’s instructions at a speed of 800 rpm and a torque of 1 Ncm. The instrument was placed in a contra- angle hand piece and was removed from the plastic tube in rotation mode by applying a lateral movement. With no rotation, the XP-Endo Finisher file was placed into the canal. Subsequently, rotation was started, and the instrument was activated for 1 min using slow and gentle 7-8 mm lengthwise movements up to the WL.The instrument was used in a brushing action against the root canal walls. All procedures with the XP-Endo Finisher files were performed at 37°C inside an incubator. Irrigation was performed using 2.5% NaOCl with 1ml volume with each instrument during the procedures. The irrigation solutions were also warmed and kept at 37°C in a water bath. This was done to simulate the body temperature and create appropriate conditions for the XP-Endo Finisher instruments to undergo the phase change.Retreatment was completed with D-Race files as described in group 2 and then as in group 3. XP-Endo Finisher R file was used as supplementary file following the retreatment procedures. -4 g  with an accuracy of 10-4 g . After c-4 g .Throughout the retreatment procedure, the tooth was isolated with a rubber dam to stop the operator from seeing the root and to avoid irrigant seepage in the hole as shown in -4 accuracy  at P≤0.05 level of significance. The Kolmogorov-Smirnov and Shapiro-Wilk tests showed that data were normally distributed. Therefore, One-way ANOVA followed by Tukey’s post hoc tests and the Paired sample t-test were used to compare between different groups.As shown in Regarding the operating time, (D-Race) group was the fastest with a statistically significance among the tested groups. (D-Race+XP-Endo Finisher R) group demonstrated no statistical significance between groups (D-Race+XP-Endo Finisher) and (D-Race+XP-Endo Finisher R) groups. (G.G+H) group was the slowest one with a statistical significance among them as shown in The (G.G+H) group showed the significantly greatest percentage of apically extruded debris (37.49%), followed by the D-Race+XP-Endo Finisher R then the D-Race+XP-Endo Finisher group  (P<0.001). The D-Race group resulted in significantly the least extruded debris (9.12%) (P<0.001) .Regardless of the retreatment technique used, some untouched parts of the root canal filling remain, potentially affecting the prognosis . In thisRegarding the remnants of root canal filling after retreatment, in this study, the H files group showed the greatest total amount of remaining root canal filling. Interestingly, H files were the most effective method in the removal of root canal filling from the coronal third. This finding is in accordance with a previous study which could be explained by the large H files used from #60 to #40 .D-Dace 0.04 file was used in the current study because it displayed less amount of filling remnants . D Race Although none of the supplementary approaches (XP-Endo Finisher and XP-Endo Finisher R) could completely free the root canals from filling remnants, both significantly decrease the amount of root canal filling. The physical characteristic features of the XP-Endo files, chiefly its ability to transform its shape under the body temperature and expand its action part to 6 mm of its axis, might let the file to touch the root canal walls thus increasing the removal of remnants . The actThis finding is in accordance with a previous study . It was Regarding the operating time of retreatment procedures, the findings of our study disclosed that D Race files recorded the least operating time, followed by D Race files with XP-Endo Finisher R, followed by D Race files with XP -Endo Finisher; H file was the slowest. This result could be due to plasticization of gutta-percha from rotary instrumentation, making penetration and retrieval of the softened gutta-percha much easier . D Race Organic and inorganic residues, gutta-percha, sealer, and irrigation solutions might extrude apically throughout the retreatment procedures, resulting in an undesirable consequence. . It was XP-Endo Finisher R extruded more debris than XP-Endo Finisher. This could be because XP-Endo Finisher R has a larger core diameter. Additionally, modifications in the instrument’s tip, making it harder and more forceful in retreatment procedures and more efficient in removing root canal filling, increased the debris extruded apically .Since XP-Endo finisher and XP-Endo finisher R are manufactured to be used at high temperatures, the intracanal temperature was simulated in this study. However, the absence of the simulated apical pressure of the periodontal ligament might limit this methodology .The main limitation of this study is that remnants of the filling material after retreatment procedures was not measured using the micro-CT. It is a non-invasive approach that allows for a three-dimensional quantitative evaluation of filling material and dentine removal during retreatment , 14, 24,Under the conditions of this study, the null hypothesis is rejected. XP- Endo Finisher files seemed to be more effective as supplementary files in the removal of failed gutta-percha after initial removal by D race files. XP- Endo Finisher R files were even more effective than XP- Endo Finisher files. Given the results of the present study, the complete removal of filling material during orthograde root canal retreatment remains a challenge. Thus, the development of new techniques, instruments, or solutions is needed to ensure complete removal of the filling materials from the root canal system. Further studies are needed to investigate the efficacy of XP-Endo Finisher R during retreatment of different obturation techniques using bioceramic sealers. Clinical studies on post-operative pain and flare-up incidence, following the use of XP- Endo Finisher and XP- Endo Finisher R during retreatment procedures, should be also conducted. Further research is also necessary to evaluate the impact of these new supplementary approaches on the outcome of root canal retreatment.Supplementary techniques are able to reduce the volume of remaining filling materials. XP-Endo Finisher and XP-Endo Finisher R significantly increased the cleaning ability when used as supplementary approaches in endodontic retreatment. However, the XP-Endo Finisher R cleaned canals significantly more than the XP-Endo Finisher and extruded more debris apically. H file was the least effective in cleaning root canals during retreatment. D Race files were the fastest technique in retreatment among all groups.